Synthesis of the new mannosidase inhibitors, diversity-oriented 5-substituted swainsonine analogues, via stereoselective mannich reaction

Article abstract of DOI:10.1021/ol049947m

5α-Substituted swainsonine analogues were synthesized by Mannich reaction of an in situ generated (-)-swainsonine iminium ion intermediate. 5α-Substituted swainsonine analogues were epimerized to their 5β-isomers in protic solvent.

Full text of DOI:10.1021/ol049947m

ORGANIC
LETTERS
2004
Vol. 6, No. 5
827-830
Synthesis of the New Mannosidase
Inhibitors, Diversity-Oriented
5-Substituted Swainsonine Analogues,
via Stereoselective Mannich Reaction
,†
Tomoya Fujita,† Hideko Nagasawa,* Yoshihiro Uto,† Toshihiro Hashimoto,‡
Yoshinori Asakawa,‡ and Hitoshi Hori†
Department of Biological Science and Technology, Faculty of Engineering,
The UniVersity of Tokushima, Minamijosanjimacho-2, Tokushima 770-8506 Japan,
and Faculty of Pharmaceutical Sciences, Tokushima Bunri UniVersity, 180
Nishihamabouji, Yamashiro-cho, Tokushima 770-8514 Japan
nagasawa@bio.tokushima-u.ac.jp
Received January 9, 2004
ABSTRACT
5r-Substituted swainsonine analogues were synthesized by Mannich reaction of an in situ generated (−)-swainsonine iminium ion intermediate.
5r-Substituted swainsonine analogues were epimerized to their 5â-isomers in protic solvent.
Golgi R-mannosidase II (GMII), a key enzyme in the
biosynthesis of N-linked glycoproteins, is a molecular target
for anticancer agents.1 The distribution of the N-linked sugars
on the cell surface is altered in various tumor cell lines, and
this unusual protein glycosylation correlates with the pro-
gression of tumor metastasis.2 In preliminary clinical trials,
(-)-swainsonine (1), a potent inhibitor of GMII, was found
to reduce tumor growth and metastasis.3 It has also been
shown to exhibit pleiotropic effects as an immunomodulatory
agent. These effects include augmentation of tumoricidal
activities of natural killer cells4 and macrophages,5 as well
as stimulation of bone marrow cells.6
Owing to such biological properties, many molecular
modifications of swainsonine (1) (summarized in Figure 1)
have been made and studied in efforts to gain insight on the
molecular bases of these activities as well as to develop more
potent immunomodulatory anticancer agents.7 For example,
Pearson synthesized the first analogues of swainsonine bear-
ing carbohydrate-like R-substituents at C(3).7e These were
shown to be more potent inhibitors of jack bean R-mannosi-
(4) (a) Galustian, C.; Foulds, S.; Dye, J. F.; Guillou, P. J. Immunophar-
macol. 1994, 27, 165. (b) Fujieda, S.; Noda, I.; Saito, H.; Hoshino, T.;
Yagita, M. Arch. Otolaryngol. Head Neck Surg. 1994, 120, 389.
(5) (a) Das, P. C.; Roberts, J. D.; White, S. L.; Olden, K. Oncol. Res.
1995, 7, 425. (b) Grzegorzewski, K.; Newton, S. A.; Akiyama, S. K.;
Sharrow, S.; Olden, K.; White, S. L. Cancer Commun. 1989, 1, 373.
(6) Oredipe, O. A.; Furbert-Harris, P. M.; Green, W. R.; White, S. L.;
Olden, K.; Laniyan, I.; Parish-Gause, D.; Vaughn, T.; Griffin, W. M.;
Sridhar, R. Pharmacol. Res. 2003, 47, 69.
(7) (a) Michael, J. P. Nat. Prod. Rep. 2003, 20, 458. (b) Asano, N.; Kato,
A.; Watson, A. A. Mini ReV. Med. Chem. 2001, 1, 145. (c) El Nemr, A.
Tetrahedron 2000, 56, 8579. (d) Pearson, W. H.; Hembre, E. J. Book of
Abstracts. In 213th ACS National Meeting; San Francisco, April 13-17,
1997; American Chemical Society: Washington, DC, 1997; ORGN. (e)
Pearson, W. H.; Guo, L. Tetrahedron Lett. 2001, 42, 8267. (f) Carmona,
A. T.; Fuentes, J.; Robina, I.; Rodriguez Garcia, E.; Demange, R.; Vogel,
P.; Winters, A. L. J. Org. Chem. 2003, 68, 3874.
† The University of Tokushima.
‡ Tokushima Bunri University.
(1) (a) Goss, P. E.; Baker, M. A.; Carver, J. P.; Dennis, J. W. Clin. Cancer
Res. 1995, 1, 935. (b) Roberts, J. D.; Klein, J. L.; Palmantier, R.; Dhume,
S. T.; George, M. D.; Olden, K. Cancer Detect. PreV. 1998, 22, 455.
(2) (a) Dennis, J. W.; Granovsky, M.; Warren, C. E. Bioessays 1999,
21, 412. (b) Dennis, J. W.; Granovsky, M.; Warren, C. E. Biochim. Biophys.
Acta 1999, 1473, 21.
(3) (a) Goss, P. E.; Reid, C. L.; Bailey, D.; Dennis, J. W. Clin. Cancer
Res. 1997, 3, 1077. (b) Goss, P. E.; Baptiste, J.; Fernandes, B.; Baker, M.;
Dennis, J. W. Cancer Res. 1994, 54, 145.
10.1021/ol049947m CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/10/2004

Scheme 2
Figure 1. Molecular modifications of (-)-swainsonine (1).
dase than swainsonine itself. The X-ray crystallographic
structure of GMII in the presence of swainsonine supported
his proposal that the 3-substituent is an effect mimic of a
disaccharide.8,9 Introduction of 6- or 7-substituents in either
axial and equatorial orientations resulted in loss of activity.10
These molecular modifications were viewed as a key to a
diversity-oriented synthetic strategy.11 The goals were to
optimize the binding interactions between drug and target
macromolecule and to delineate the disease-related biological
pathways.
methylation, and then palladium-catalyzed hydrogenolysis
to give the cyclic amine acetal 5 in good yields.
Acid-catalyzed cleavage of protective groups present in
amine acetal 5 in protic solvent followed by treatment with
anion-exchange resin (Dowex 1X8) did not produce the
desired iminium salt. Instead, a swainsonine dimer 8 was
formed by reaction of iminium ion 6 with the corresponding
enamine 7 (Scheme 2) (Table 1, entries 1 and 2).
As part of our contributions to the development of new
swainsonine analogues to help achieve these goals, we
describe herein the stereoselective synthesis of 5-substituted
swainsonine analogues and the structure-activity relationship
(SAR) of their R-mannosidase inhibitory activities. To
introduce substituents to (-)-swainsonine (1) in the 5-posi-
tion, a Mannich reaction was performed with a swainsonine
iminium ion generated in situ from amine acetal 5. As shown
in Scheme 1, the key iminium salt precursor 5 was
Table 1. Solvent Effect on Dimerization Reaction
yielda (%)
entry
acid
HCl
HCl
HCl
solvent
time (h)
8
9
1
2
3
4
H2O
21.5
24
1.5
24
quant
68b
7b
0
MeOH
CH3CN
CH3CN
33b
92b
81
BF3‚Et2O
0
1
a Isolated yields. b Determined by H NMR.
Scheme 1
In contrast, the intramolecular acetal 9 was obtained as a
major product if deprotection were carried out in acetonitrile
as a solvent (Table 1, entries 3 and 4). The iminium salt 6
apparently is not so readily formed in acetonitrile because
the nitrogen lone pair is less available under the aprotic acidic
conditions that stabilize cationic species, than in protic
solvents. However, intramolecular acetal 9 could be con-
verted into the desired iminium salt 6 under these conditions
since the ratio of 8 to 9 increased from 68/33 (Table 1, entry
2) to 87/4 after and additional 66 h reaction time.
1
With respect to dimer formation, H NMR analysis of 8
revealed that enamine 7 attacked the iminium salt 6 from an
equatorial direction to afford the more stable adduct 8
selectively. The chemical shift for H-5 was observed at δ
2.24 (dd, J ) 2.1, 11.3 Hz) in swainsonine dimer 8. The
large J value (11.3 Hz) between H-5 and H-6 was also
indicative of trans diaxial coupling and equatorial orientation
(â-position) of the swainsonine enamine moiety, the vinyl
synthesized from azido lactone 3,12 which was prepared from
commercially available 2,3-O-isopropylidene-D-erythrono-
lactone (2) as described in the literature.13 The azido lactone
3 was subjected to reduction with diisobutylalumium hydride,
(8) van den Elsen, J. M.; Kuntz, D. A.; Rose, D. R. EMBO J. 2001, 20,
3008.
(9) Numao, S.; Kuntz, D. A.; Withers, S. G.; Rose, D. R. J. Biol. Chem.
2003, 278, 48074.
(10) Pearson, W. H.; Hembre, E. J. Tetrahedron Lett. 2001, 42, 8273.
(11) Schreiber, S. L. Science 2000, 287, 1964.
(12) Selected data for 3: [R]22D +78.6 (c 0.51, CHCl3); mp 140 °C (from
CHCl3/Et2O) [lit.13 [R]23D +75.0 (c 0.52, CHCl3); mp 136 °C (from CHCl3/
Et2O)]. 3 was converted to (-)-swainsonine ([R]24D -86.9 (c 0.13, MeOH)
[lit.13 [R]23D -74.0 (c 0.98, MeOH), lit.17[R]20D -87.2 (c 1, MeOH)] with
an overall yield of 46% according to ref 13.
(13) Pearson, W. H.; Hembre, E. J. J. Org. Chem. 1996, 61, 7217.
828
Org. Lett., Vol. 6, No. 5, 2004

Table 2. Mannich Reaction of Amine Acetal 5 and Various
Ketones
yield (%)
entry
R
solvent
EtOH
EtOH
80% CH3CN/ 70
EtOH
T (°C) time h
10
8
1a
2b
3c
Phd
Ph
rt
reflux
28
1
a
a
a
46
38
72
53
42
30
Phd
3
4a
5b
p-MePhd EtOH
p-t-BuPh EtOH
p-BrPh
p-IPh
Et
70
2
2
3
0.5
4
11.5
32
32
b
c
d
e
f
g
h
i
49
34
29
18
28
32
31
29
22
32
36
55
61
50
23
4
reflux
reflux
reflux
reflux
reflux
reflux
reflux
reflux
6b
EtOH
EtOH
MeOH
MeOH
MeOH
EtOH
MeOH
7b
8b
Figure 2. Perspective view and stereoview of tri-4-bromobenzoate
of 10b.
9b
Pr
10b
11b
12b
n-Bu
Cy
t-Bu
36
58
19
j
improved the yield of Mannich product (Table 2, entry 3),
attempted reaction in pure acetonitrile led only to decom-
position of starting material. Aromatic ketones were more
reactive than aliphatic ones presumably due to greater
stabilization of their enol form (Table 2, entries 1-5).
Unsymmetrical dialkyl ketones gave a single regioisomer by
reaction at the less substituted R carbon (Table 2, entries
8-11).16 It was interesting to note that the less stable
diastereomers resulting from axial attack (R-position) of enols
were formed selectively. This result is in contrast to the
formation of the swainsonine dimer byproduct 8 that occurs
through equatorial attack. The stereochemistries of Mannich
adducts were determined by their NMR analyses and X-ray
diffraction. The Mannich adduct 10b [(5R)-5-[2′-oxo-2′-(4-
methylphenyl)ethyl]swainsonine] was converted to the 4-bro-
mobenzoylate to give a single crystal for X-ray determination
of the absolute stereochemistry (Figure 2). Its X-ray structure
confirmed that the 5-substituent occupied the axial position
and that the ring junction was trans-fused. In all adducts,
the chemical shift for H-5 appeared in the 3.62-3.35 ppm
range (Table 3) and a Bohlmann band (2800-2850 cm-1)
was present in the IR spectrum. These data indicated that
products were formed exclusively by axial attack. Axial
attack on the cyclic iminium salt in a half-chair conformation
through a stable chairlike transition state would be favored
under conditions of kinetic control.
a HCl (3 equiv). b 11 equiv. c 1.5 equiv. d When the SM spot disappeared
on TLC, 6 N HCl was added and the solution was stirred for 30 min at 70
°C.
proton H-5′ of which appeared at the lowest field (δ 5.97).
The large J value (9.2 Hz) between H-8a (δ 1.75, dd, J )
3.8, 9.2 Hz) and H-8 (δ 3.76, ddd, J ) 4.8, 9.2, 10.9 Hz)
also indicated that they were in a trans diaxial relationship.
The axial orientation of the bridgehead proton H-8a and a
Bohlmann band (2854 cm-1) in the IR spectrum were
consistent with the trans-fused indolizidine structure.14
Formation of this trans-fused indolizidine dimer indicated
that stereoselective acid-catalyzed Mannich reactions of
iminium salt 6 derived from 5 would occur in protic solvent
to produce 5-substituted swainsonine analogues. We next
examined reactions of amine acetal 5 and various ketones
(Table 2) in order to introduce various alkyl groups at C(5)
of swainsonine. 15 In all reactions, two major fractions were
obtained, as shown by reversed-phase HPLC. The less polar
product proved to be the Mannich adduct. The more polar
product was not identified, but this same byproduct was
obtained in every reaction with various ketones. Upon being
passed through a basic ion-exchange column (Dowex 1X8),
this byproduct was converted to swainsonine dimer 8.
Whereas the addition of acetonitrile to protic solvent
We also found that it is possible to epimerize the
stereocenter at C(5) to afford the (5â)-diastereomers of the
Mannich adducts. This can be effected in reasonable yield
by incubation in MeOH for several days or by the treatment
with basic ion-exchange resin (Dowex 1X8) in 50% MeOH/
water for several hours (Scheme 3). After this inversion to
(14) (a) Bohlmann, F.; Mueller, H. J.; Schumann, D. Chem. Ber. 1973,
106, 3026. (b) Bohlmann, F.; Schumann, D.; Arndt, C. Tetrahedron Lett.
1965, 2705.
(15) General Procedure. A solution of 5 (10 mg, 0.04 mmol) in a solvent
(500 µL) was added to a solution of 40 to 100% excess of various ketones
(250-500 µL) and concentrated HCl in a solvent (500 µL) under reflux.
After the reaction for the period of time indicated in Table 2, the mixture
was evaporated, dissolved in water and extracted with Et2O. The aqueous
layer was lyophilized and purified by preparative reversed-phase HPLC
(C18, acetonitrile/water gradient elution system from 0 to 100% over the
period of 60 min).
(16) Jasor, Y.; Gaudry, M.; Luche, M. J.; Marquet, A. Tetrahedron 1977,
33, 295.
Org. Lett., Vol. 6, No. 5, 2004
829

Scheme 3a
Table 3. NMR Chemical Shiftsa (H-5) and R-Mannosidase
Inhibitory Activityb of 5-Substiutued Swainsonine Analogues
5R analogue
5â analogue
H-5eq
(ppm)
IC50
H-5ax
(ppm)
IC50
R
(µM)
(µM)
Ph
10a
10b
10c
10d
10e
10h
10i
3.62
3.59
3.51
3.50
3.51
3.45
3.35
3.48
0.51
0.25
0.22
0.42
0.33
1.27
0.82
2.45
11a
11b
11c
11d
11e
11h
11i
2.54
2.62
2.52
2.67
2.54
2.40
2.31
NDc
2.24
1.55
0.91
2.54
NDc
NDc
2.70
3.29
NDc
3.80
a Key: (a) Dowex 1X8 OH- form, 50% MeOH/H2O; (b) MeOH.
p-MePh
p-t-BuPh
p-BrPh
p-IPh
n-Bu
subsutituent on the (5R)-face of swainsonine. This is sup-
ported by the crystal structure of GMII in the presence of
swainsonine that shows C(3) and C(5) of swainsonine located
at the entrance of the binding pocket.8
Cy
t-Bu
10j
11j
Dimer 8
In summary, we have developed amine acetal 5 as a
versatile intermediate for the preparation of 5R- and 5â-
substituted swainsonine analogues by stereoselective Man-
nich reaction and epimerization. The new 5R-substituted
swainsonine analogue 10c prepared by this route was found
to be a more potent R-mannosidase inhibitor than swainso-
nine itself. Using the present procedure and other approaches,
we are exploring greater diversity of C(5) substituted
swainsonine analogues to study the SAR of swainsonine with
respect to GMII inhibitor and immunomodulatory anticancer
activity.
a δ values in ppm from TMS. b The IC50 for inhibition of jack bean
R-mannosidase, IC50 of (-)-swainsonine; 0.25 µM, (lit.18 0.4 µM). c ND:
not determined.
the â configuration, H-5 protons exhibited a upfield shift to
2.67-2.31 ppm in the 1H NMR spectra. These relationships
are similar to that between axial proton H-5ax (δ 1.85) and
equatorial proton H-5eq (δ 2.90) of swainsonine.17
Mannosidase inhibitory activities of the new 5-substituted
swainsonine analogues were evaluated against jack bean
R-mannosidase.18 All of the (5R)-substituted swainsonine
analogues have higher inhibitory activity than the corre-
sponding (5â) epimer (Table 3). The axial-oriented (R-face)
aromatic substituents at C(5) position proved to be more
effective for inhibitory activity than aliphatic groups. In-
creasing the size of a para-subsutituent of the phenyl ketone
group on the R-face of swainsonine also resulted in an
increase in the activity. Analogue 10c, with an R-oriented
2-oxo-2-(4-tert-butylphenyl)ethyl substituent, and 10b, with
an R-oriented 2-oxo-2-(4-methylphenyl)ethyl substituent,
compared favorably with swainsonine. On the other hand,
bulky groups on the â-face tended to diminish potency. These
data suggest that the substrate or inhibitor binding site of
R-mannosidase afforded enough space for an aromatic
Acknowledgment. We are grateful to Dr. S. Takaoka
(Tokushima Bunri University) for X-ray crystal structure
analysis. We also thank M. Nakamura, E. Okayama, and K.
Yamashita of our faculty for excellent NMR spectral,
elemental analysis, and mass spectral techniques, respec-
tively. We thank Dr. Kenneth L. Kirk for his helpful
comments and valuable suggestions.
Note Added after ASAP Posting. The IC50 values in Table
3 were incorrect in the version posted February 10, 2004;
the corrected version was posted February 12, 2004.
Supporting Information Available: Spectral data for all
new compounds and X-ray crystallographic data of tri-4-
bromobenzoate of 10b. This material is available free of
charge via the Internet at http://pubs.acs.org.
(17) Kardono, L. B. S.; Kinghorn, A. D.; Molyneux, R. J. Phytochem.
Anal. 1991, 2, 120.
(18) Dennis, J. W.; White, S. L.; Freer, A. M.; Dime, D. Biochem.
Pharmacol. 1993, 46, 1459.
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Org. Lett., Vol. 6, No. 5, 2004